Successful slush nitrogen vitrification of human ovarian tissue

The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification of human ovarian cortical strips with slush nitrogen compared with liquid nitrogen.

Riccardo Talevi, Ph.D., Vincenza Barbato, Ph.D., Ilaria Fiorentino, Ph.D., Sabrina Braun, Ph.D., Cristofaro De Stefano, M.D., Raffaele Ferraro, M.D., Sam Sudhakaran, M.Sc., Roberto Gualtieri, Ph.D.

Volume 105, Issue 6, Pages 1523-1531


To study whether slush nitrogen vitrification improves the preservation of human ovarian tissue.

Control vs. treatment study.

University research laboratory.

Ovarian biopsies collected from nine women (aged 14–35 years) during laparoscopic surgery for benign gynecologic conditions.


Main Outcome Measure(s):
Ovarian cortical strips of 2 × 5 × 1 mm were vitrified with liquid or slush nitrogen. Fresh and vitrified cortical strips were analyzed for cryodamage and viability under light, confocal, and transmission electron microscopy.

Compared with liquid nitrogen, vitrification with slush nitrogen preserves [1] follicle quality (grade 1 follicles: fresh control, 50%; liquid nitrogen, 27%; slush nitrogen, 48%); [2] granulosa cell ultrastructure (intact cells: fresh control, 92%; liquid nitrogen, 45%; slush nitrogen, 73%), stromal cell ultrastructure (intact cells: fresh control, 59.8%; liquid nitrogen, 24%; slush nitrogen, 48.7%), and DNA integrity (TUNEL-positive cells: fresh control, 0.5%; liquid nitrogen, 2.3%; slush nitrogen, 0.4%); and [3] oocyte, granulosa, and stromal cell viability (oocyte: fresh control, 90%; liquid nitrogen, 63%; slush nitrogen, 87%; granulosa cells: fresh control, 93%; liquid nitrogen, 53%; slush nitrogen, 81%; stromal cells: fresh control, 63%; liquid nitrogen, 30%; slush nitrogen, 52%).

The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification with slush nitrogen compared with liquid nitrogen.

  • Elham Amini

    I am an anatomical pathologist in Perth, Australia. I am interested to take part in a similar study to yours. I would appreciate it if you tell me about the details of how you processed the samples for histology, such as how long did you fix samples in formalin, both the control and the tissue after 24 hours culture.

    Thank you very much and kind regards

    Elham Amini

    Fellow of the Royal college of Australia FRCPA

    • Riccardo Talevi

      dear Elham AminiWe fix all samples in bouin fixative for 4 hours Then Wash briefly in Water And dehydrated through passages in crescent concentrations of ethanol, particularly 1h in 50%, at least 3h in 75%,overnight in 95%,3x1h in 100% And finally embedding in paraffin

      • Elham Amini

        Dear Riccardo
        We are having our first patient for the ovarian vitrification study, and we are using your study as our guideline. I would appreciate it if you could give me more information about the details of how many H&E sections and with what intervals (the details of your protocol) you prepared for each patient and how many slides you ended up with for each patient in the study?
        Many thanks and kind regards

  • Amir Arav

    Dear Dr. Talevi

    These is very good paper, however you forgot to described how and who was the first to apply LN slush in ART.
    Selected publication on LN slush

    1. Arav, A., Yavin, S., Zeron, Y., Natan, Y., Dekel, I., Gacitua, H.(2002) New trend in gamete’s cryopreservation.Mol. Cell. Endocrinology 187, 77-81
    2. Yavin S., Arav A., (2007) (Review) Measurement of essential physical properties of vitrification solutions. Theriogenology. 2007 Jan 1;67(1):81-9
    3. Yavin S, Aroyo A, Roth Z, Arav A.(2009) Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush. Hum Reprod.;24(4):797-804.
    4. Saragusty J, Arav A. Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification. Reproduction. 2011 Jan;141(1):1-19
    5. Arav A. Cryopreservation of oocytes and embryos. Theriogenology. 2014 Jan 1;81(1):96-102. Review

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